Polypeptides can adopt three-dimensional structures that are capable of binding to other biological molecules with very high affinity and specificity. A library of random polypeptide sequences can be populated by molecules with a wide variety of three-dimensional structures. In order to isolate a polypeptide with a conformation that interacts with a specific target protein, individual sequences from the library can be prepared and tested or screened for their affinity to the target. However, for very large libraries (>106 members), the screening of individual sequences for binding affinity is not feasible. To overcome this limitation, a number of techniques have been developed to select novel polypeptides from extremely large, complex mixtures by virtue of their binding affinity to a target.
The ribosome contains two sites that are critical for the function of aminoacylated tRNAs in protein synthesis: the peptidyl transferase center and the decoding site region. The mRNA display strategy developed by Szostak et al. (e.g., U.S. Pat. No. 6,258,558; and Roberts, R. W. and Szostak, J. W. (1997). Proc. Natl. Acad. Sci. USA 94:12297-12302) exploits the catalytic activity of the peptidyl transferase center to link a 3′-puromycin derivatized mRNA to its encoded peptide by creating a peptide bond between the two.
An mRNA can also be linked to a tRNA at the decoding center. This can be achieved through the use of naturally-occurring or artificially-introduced crosslinkers. One naturally-occurring photochemical crosslinker is the well-known Y-base present near (within a few angstroms of) the anticodon of certain tRNAs (see, e.g., U.S. Pat. Nos. 5,843,701; 6,194,550; and 6,440,695). Others (e.g., U.S. Pat. Nos. 6,962,781; 7,351,812; 7,410,761; and 7,488,600) have described combinations wherein a photo-activatable group on one nucleic acid can react with a reactive group on a second nucleic acid.